Soybean is an agronomically important crop. Despite this importance, however, there is only limited breeding potential in the United States due to a small germplasm base. As a result, tremendous efforts have been expended in developing techniques to modify soybean characteristics through the use of genetic engineering. Such modifications offer the possibility of developing plant lines that have specific, tailor-made beneficial traits, such as herbicide resistance, drought resistance, heat resistance, disease resistance, seed quality improvement, and the like, in ways not possible using traditional breeding techniques. Due to the successes of Agrobacterium tumefaciens-mediated gene transfer in other plant species, much effort has been placed on developing such a system for the genetic modification of soybean. To date, however, these efforts have met with only limited success, with generally low transformation efficiencies on most soybean cultivars.
Agrobacterium tumefaciens is a gram-negative soil bacteria that causes the crown gall disease in plants by infecting cells through wound sites. A. tumefaciens infects by injecting into the cell a strand of DNA (termed T-DNA) derived from the large tumor-inducing (Ti) plasmid (van Larebeke et al., Nature 255: 742-743, 1975). The T-DNA then integrates into a chromosomal location in the plant and produces enzymes that synthesize hormones which cause the crown gall symptoms (Chilton et al., Cell 11: 263-271, 1977). The genes encoding these enzymes, and the eukaryotic regulatory control elements associated therewith, are located on the T-DNA. In addition, the integrated T-DNA also encodes products that direct the synthesis of compounds known as opines, which are amino acid and sugar derivatives, which varies depending upon the A. tumefaciens strain.
Mobilization of the T-DNA requires that the products of genes located elsewhere on the Ti plasmid, called collectively the vir genes, which are activated by certain elicitors from the wounded plant cells in trans to synthesize and transfer a single-stranded copy of the T-DNA (the T-strand) to the plant cell (Zambryski, Ann. Rev. Plant. Physiol. Plant Mol. Biol. 43: 465-490, 1992; Zupan and Zambryski, Plant Physiol. 107: 1041-1047, 1995). The T-DNA sequence on the Ti plasmid is flanked by short 24-bp direct repeats (Yadav et al., Proc. Natl. Acad. Sci. (USA), 1982), which are required for the recognition of the T-DNA (Wang et al., Cell 38: 455-462, 1984). Sequences immediately surrounding these borders appear to be involved in the polarity of T-strand synthesis, which initiates at the right border (Wang et al., Mol. Gen. Genet. 210: 338-346, 1987).
The discovery of the mechanism by which A. tumefaciens infects plant cells, i.e. by DNA transfer, led to the realization that this microorganism might be useful, via its Ti plasmid, for transferring agronomically useful genes to plants. Recently it has been demonstrated that foreign DNA, flanked by T-DNA border sequences, can be transferred into plant cells using A. tumefaciens as the vector (Hernalsteens et al., Nature 287:654-656, 1980). Furthermore, inactivation or removal of the native T-DNA genes involved in hormone synthesis would render the A. tumefaciens incapable of producing the crown gall disease symptoms. This process of inactivating or removing genes responsible for disease symptoms is termed "disarming."
The first methods of A. tumefaciens engineering involved the simultaneous disarming and introduction of the desired gene, since the introduced gene directly replaced the genes in the T-DNA. By a method termed "homogenotization" (Matzke and Chilton, J. Mol. Appl. Genet. 1: 39-49, 1981), the native T-DNA of the Ti plasmid was replaced with a desired gene for transformation. Homologous recombination occurred between the T-DNA of the Ti plasmid and an intermediate construct in a broad host range plasmid, containing the desired gene and a selectable marker (e.g., drug resistance) flanked by T-DNA sequences. The recombination event was forced by a subsequent introduction of a second broad host range plasmid incompatible to the intermediate construct, and selecting for drug-resistance encoded by the selectable marker gene of the introduced T-DNA in the desired construct, and the drug-resistance gene on the incompatible plasmid.
Another strategy developed for engineering A. tumefaciens involved cloning the desired gene into a cointegrative intermediate vector, which contained a single region of T-DNA homology and a single border sequence. In this system, the sequences are recombined by a single-crossover event (Horsch et al., Science 227: 1229-1231, 1985), which results in the entire vector, including the gene of interest, being integrated. Cointegrative systems pair in regions of homology between the T-DNA region of the Ti plasmid and the DNA sequence on the introduced integrative vector.
One example of a useful cointegrative plasmid is pGV3850, a Ti plasmid from a nopaline strain (C58), from which the entire T-DNA region between the borders was replaced with pBR322, thus offering a recombination site for any gene construct containing pBR322 homology (Zambryski et al., EMBO J. 2(12): 2143-2150, 1983).
Upon the discovery that T-DNA does not have to be on the same plasmid as the vir genes (de Framond et al., Bio/Technol. 1: 262-269, 1983; Hoekema et al., Nature 303: 179-180, 1983), the binary vector was developed. A binary vector is maintained in the A. tumefaciens separate from the Ti plasmid, and contains the gene of interest and a selectable marker gene between T-DNA border sequences. These vectors offer a great degree of flexibility, since they do not require a specifically engineered Ti plasmid with a homologous recombination site. For that reason, any disarmed A. tumefaciens strain can be used to transfer genes for any binary vector. Owing to their versatility, binary vectors are currently the preferred intermediate vectors for cloning genes destined for A. tumefaciens-mediated transfer into plants. However, any A. tumefaciens strain to be used with binary vectors must have its own Ti plasmid disarmed, especially if the target plant species is inefficiently transformed via A. tumefaciens. Otherwise, the desired gene from the binary vector will be co-transformed with the oncogenic phytohormone genes from the native T-DNA of the bacteria, thereby reducing transformation efficiency of the desired gene and also producing the tumorigenic disease symptoms in many of the target cells and thereby preventing the differentiation of these cells into normal plants.
Disarming wild-type A. tumefaciens strains for general use with binary vectors has involved, in some cases, a form of homogenotization. An intermediate construct containing a marker gene flanked by Ti plasmid sequences that are homologous to regions that lie outside the T-DNA, is introduced into the wild-type A. tumefaciens by bacterial conjugation (Hood et al., J. Bacteriol. 168(3): 1291-1301, 1986; Hood et al., Transgenic Res. 2: 208-218, 1993). Whereas disarmed A. tumefaciens strains typically have their entire T-DNA sequences removed, it has also been demonstrated that T-DNA mobilization can be inactivated by removal of the right border sequence: reports from work with nopaline-type strains of A. tumefaciens show that the right border of T-DNA is necessary for gene transfer, whereas the left border is not. (Joos et al., Cell 32: 1057-1067, 1983; Peralto and Ream, Proc. Natl. Acad. Sci. (USA), 1985; Shaw et al., Nucleic Acids Res., 12: 6031-6041, 1984; Wang et al., Mol. Gen. Genet. 210: 338-346, 1984).
A. tumefaciens has a diverse dicot host range, and additionally some monocot families (De Cleene and De Layk, Bot. Rev. 42 (4): 389-466, 1976). There are several different strains of A. tumefaciens, each classified into octopine-type, nopaline-type, and L,L-succinamopine-type, named after type of opine synthesized in the plant cells they infect. These strains have comparable, although not identical, host ranges and disarmed versions of many types of A. tumefaciens have been used successfully for gene transfer into a variety of plant species. (van Wordragen et al., Plant Mol. Biol. Rep. 10: 12-36, 1992; Hood et al., Transgenic Res. 2: 208-218, 1993). Although its most sensitive hosts are members of the dicot family Solanaceae, A. tumefaciens, as mentioned above, has also been demonstrated to infect some monocots as well (Smith and Hood, Crop Sci. 35(2): 301-309, 1995).
However, soybean (Glycine max L. Merr.) has proven to be very difficult to transform with A. tumefaciens, at least in part because it is refractory to infection by wild-type A. tumefaciens. Comparative studies with a number of soybean cultivars and A. tumefaciens strains suggest that soybean susceptibility to A. tumefaciens is limited, and is both cultivar- and bacterial strain dependent (Bush and Pueppke, Appl. Environ. Microbiol. 57(9): 2468-2472, 1991; Byrne et al., Plant Cell Tiss. Org. Cult. 8: 3-15, 1987; Hood et al., Plant Physiol. 83:529-534, 1987). The problems with soybean recalcitrance to A. tumefaciens are further complicated by the difficulty of working with soybean in tissue culture.
Progress in A. tumefaciens-mediated gene transfer in soybean is limited by two major factors: (1) development of a soybean tissue culture system that efficiently regenerates plants from a single-cell origin (Cheng et al., Plant Sci. Let. 19: 91-99, 1980; Wright et al., Plant Cell Rep. 5: 150-154, 1986), and (2) further understanding of the mechanism for A. tumefaciens-mediated gene transfer, i.e., the fact that certain chemical elicitors applied externally (e.g., acetosyringone) can stimulate vir gene activation and T-DNA transfer into cells of non-host plants such as soybean (Owens and Smigocki, Plant Physiol. 88: 570-573, 1988; Stachel et al., Nature 318: 624-629, 1985).
One system for soybean A. tumefaciens-mediated gene transfer has now been established, and is in wide use (Townsend, International Patent Application WO 94/02620, 1994; Hinchee and Conner-Ward, U.S. Pat. No. 5,416,011, 1995). Despite these advances to date, however, A. tumefaciens-mediated gene transfer into soybean remains inefficient and labor-intensive, and methods for improving that efficiency are continually being sought.
As mentioned earlier, some A. tumefaciens strains infect soybean more readily than others. One strain, A281, is a supervirulent, broad host-range, L,L-succinamopine-type A. tumefaciens strain that shows high virulence on soybean. Strain A281 has a nopaline-type C58 chromosomal background, containing the L,L-succinamopine-type Ti plasmid, pTiBo542, and out-performs its chromosomal and Ti plasmid progenitors on soybean (Hood et al., Plant Physiol. 83: 529-534, 1987). Disarming this strain has produced EHA101 and EHA105, strains now widely used in conjunction with soybean transformation (Hood et al., J. Bacteriol. 168(3): 1283-1290, 1986; Hood et al., Plant Physiol. 83: 529-534, 1987).
Recently, Chry5, another L,L-succinamopine-type strain of A. tumefaciens recovered from chrysanthemum, has been found to have a broad host range, and is also highly virulent on soybean (Bush and Pueppke, Appl. Environm. Microbiol. 57(9): 2468-2472, 1991). The Ti plasmid of this strain, designated pTiChry5, is comparable in arrangement and homology to pTiBo542 in supervirulent strain A281. The Chry5 strain also possesses a cryptic plasmid, the purpose of which is unclear. Based on stem inoculation assays, Chry5 rivals A281 (Hood et al., Plant Physiol. 83: 529-534, 1987) for tumorigenicity on soybean.
The sequences of pTiChry5 have been subcloned as partial EcoRI fragments into a cosmid library in pLAFR1 (Friedman et al., Gene 18: 289-296, 1982), and mapped for EcoRI and BamHI sites and for vir, inc, L,L-succinamopine utilization, and the T-DNA regions, based on homology to pTiBo542 of A281. Observations from complementation analysis suggest key cis-acting elements in pTiChry5 near the T-DNA right border as being involved with supervirulence. However, studies involving transferring pTiChry5 into other A. tumefaciens strains suggest that there may also be chromosomal involvement in the hypervirulence of Chry5 observed with soybean (Kovacs and Pueppke, Mol. Gen. Genet. 242: 327-336, 1994).
U.S. Pat. No. 5,416,011 discloses a method for the transformation of soybean, with a disarmed strain of A. tumefaciens designated A208. The .beta.-glucuronidase gene under the control of the cauliflower mosaic virus (CMV) 35S promoter is disclosed as being useful for the determination of transformation efficiency.
International Patent Application WO 94/02620 discloses transformation of soybean using a disarmed A. tumefaciens designated LBA 4404 and the induction of bacterial virulence by culture in media having a pH below 6.0.
Kovacs and Peuppke (Mol. Gen. Genet. 242: 327-336, 1994) discloses the genomic organization and restriction endonuclease mapping of the Ti plasmid pTiChry5. That publication further discloses that A. tumefaciens Chry5 is a highly tumorigenic strain that has the ability to transform soybean.
Kovacs et al. (Mol. Gen. Genet. 242: 327-336, 1993) disclose that a cryptic plasmid and the bacterial chromosome of strain Chry5 potentiate the tumorigenic ability of several different Ti plasmids in comparison to their normal genetic background.
Kovacs and Peuppke (Phytopathology, 81 (10): Abstract No. 678B, 1991) briefly describes a plasmid-cured A. tumefaciens derivative of Chry5 into which the Ti plasmid of strain T37 was conjugated.
Bush and Pueppke (Appl. Environm. Micorbiol. 57(9): 2468-2472, 1991) discusses the characterization of A. tumefaciens Chry5, isolated from naturally-occurring crown galls on Chrysanthemum morifolium. Strain Chry5 is thought to be a biotype I strain that transforms at least 10 different plant species.
Hood et al. (Transgenic Res. 2:208-218, 1993) discloses the disarming of three Ti plasmids: one each of the octopine, nopaline and L,L-succinamopine types. A. tumefaciens strains A281 and EHA101 are disclosed as able to transform soybean. The disarming derivative of plasmid pTiBo542 from strain A281 is disclosed and designated pEHA105.
Hinchee et al. (Gene Manipulation in Plant Improvement II, pp. 203-212, J P Gustafson, ed., Plenum Press, New York, 1990) discusses transformation of soybean by A. tumefaciens, wherein out of 100 cultivars of soybean tested for transformation, only three were found susceptible in repeated tests. Also reported is a .beta.-glucuronidase gene marker system.
Kudirka et al. (Can. J. Genet. Cytol. 28: 808-817, 1986) discloses various characteristics of wound repair in the presence of tumorigenic and non-tumorigenic strains of A. tumefaciens and that soybean explants had recently been transformed.
From these prior disclosures, it is readily apparent that the art would significantly advance with the addition of novel strains of disarmed A. tumefaciens having a wide host-range and the ability to more efficiently transform plants, such as soybean, that have here-to-date been refractory to Agrobacterium-mediated transformation. Moreover, since A. tumefaciens strains vary somewhat in their host range, creating new disarmed strains is expected to expand the list of A. tumefaciens-transformable plant species.
It is thus one object of the invention to provide novel disarmed A. tumefaciens strains that are efficient in the transformation of economically important crops, in particular soybean. Another object of the invention is to provide novel methods for increasing the efficiency of A. tumefaciens-mediated gene transfer, again particularly with respect to soybean. In conjunction with the foregoing, it is another object of the invention to provide novel genetically engineered Ti plasmids useful in the transformation of both dicots and monocots. Yet another object of the invention is to provide novel methods for the transformation of plants, particularly soybean. A further object of the invention is to provide transgenic plants possessing one or more genetically engineered desirable characteristics. These and other objects of the invention, apparent from the disclosure herein, are realized in the A. tumefaciens strains, exemplified by strain KYRT1, under the practice of the invention.